of tebbe haute



Reissued Jan. 13, 1931 UNITED STATES PATENT. OFFICE 1 DAVID A. LEGG, 01'TEBBE HAUTE, INDIANA, ASSIGNOB TO COMMERCIAL $011M CORPORATION, OF TEBBEHAUTE, INDIANA, A CORPORATION OF MAB-ILA ART or Bum-neurone manmwrarronHo Drawing.

Original. No. 1,868,814, dated Kay 8, 1928, Serial 110. 139,016, filedOctober 1, 1928.

Application fol-reissue filed November 27, 1929. Serial No. 410,248.

My invention relates to the art of butylacetonic fermentation and hasfor its principal object the development of a reliable method ofconducting the butyl-acetonic fermentation rocess in such a manner thatgood yields 0 products are obtainable even in the presence of adversefermentation conditions.

The butyl-acetonic fermentation of carbohydrates has been carried out ona large manufacturing scale for some years past. In this process asterile mash f amylaceous or saccharine carbohydrates is prepared, andthe warm mash is inoculated with a culture of butyl-actetonic bacilli,whereupon fermentation proceeds at a temperature of about 36 (3., thisbeing the optimum fermentation temperature maintained by the bacilliwith out the outside application of heat. r

In the case of sterile corn mash of 8% concentration, a normalfermentation is complete within thirty-six to seventy-two hours, andthere is a solvent yield of about by weight of the dry corn. Thissolvent yield represents the total quantity of butyl alcohol, acetone,and ethyl alcohol obtained. The fermented mash-is then distilled in theordinary manner to remove the solvents present in the aqueous solution.

Butyl-acetonic bacilli are present in nature and may be isolated andpurified according to the method of Weizmann (U. S. Patent 1,315,585).The butyl-acetonic bacillus has previously been described by variousnames. including z-Bacillus granulobactew pectinocomm, Bacillusamylobacter, Olo'stm'dz'wm buty ricum, Bacillus butg ricus, Bacillusbutylaceticum, Bacillus acctobutg lz'cmn, Ulostri- (Hum acetobutyliaum,etc. Slight differences due to culturing methods and treatment have beenobserved but all strains have certain definatory characteristics.

The butyl-acetonic bacillus is chiefly char-u act-erized by its abilityto produce, from car-- bohydrates, a solvent mixture of but-yl alcohol(about two parts);-acetone (about one part) and, usually, smallquantities of ethyl alcohol. A mixture of hydrogen gas andcarbondioxidegas is given off during fermentation. During the first stages offerlessened until, in a normal case it is a require a preliminaryconversion of this material to sugar.

The bacillus is an anaerobe but is ordinar ily employed under practicalaerobic conditions. Young vegetative cells stain yellow with iodine, butlater; as sporulationapproaches, the cells take on elostridial form,whereupon they stain blue or violet with iodine owing to the presence ofgranulose.

Young vegetative cells are actively motile with from nine to eighteenperitrichous fiagella.- They are at rst gram-positive, but aftereighteen to twenty-four hours shrivglled grain-negative cells increasein numers.-

While a pure culture butyl-acetonic bacilli will produce a factorysolvent yield of 25% based on dry corn, or a similar figure when othercarbohydrates are fermented, the history of the art has shown recurrentperiods of lowered yields due to abnormal fermentationr There are knownin the art two distinctlydiiferent ty es of abnormal fermentation whichare or inarily classified as contaminated fermentation mentation.

. Contaminated. fermentation, as the name implies, is due to thepresence in the fermentable to prevent the appearance of contaminatingorganisms in the fermentation byproper precautions in sterilization andoperation, and 1f contaminat ng organisms do and sluggish fer-v I findtheir way into the mash it is still possible to keep them under controlor destro them by'various processes of treatment suc 'asresterilization, or' neutralization of the ly become apparent inhundreds of disconin the increasedtime required for ermentanectedvessels of various sizes, including lab oratory cultures. Even cultureswhich havebeen stored for years in sealed tubes in spore form, whentransferred to and grown in a sterile mash are not immune fromsluggishness during a plant-epidemic period.

Sluggish fermentation isprimarily char acterized by a prolonged aciditypeak and by the extreme slowness with which the end point is reached. Anepidemic of sluggishness greatly reduces the total solvents producib efrom apparatus of a given size and standard capacity in a stated time.During such an epidemic, production schedules are overthrown and greatloss ensues.

Fully as important as the factor of slowness which occurs in sluggishfermentation, is the factor of diminished yield of solvents. sluggishfermentations are rarely complete fermentations-all of the fermentablecarbohydrate is-not fermented, and an appreciable loss ensues, both inreduced solvent ield and tion.

I have now discovered a means of operating the butyl-acetonicfermentation process in a manner that prevents the loss of time and lossof solvents otherwise incurred through sluggishness, and aprocess whichgivesuniformly higher plant yields than those known in the art.

My, invention is best understood in connection with my theory for itsoperativeness though it is of course understood that my invention standsdistinct from. the theory. The recurrent and epidemic character ofsluglgishness suggests that it is a disease of t cumstances under whicha fermentation process, operating normally, first falls under theinfluence of sluggishness are not known. It is certain, however,thatwhen the mash'in one fermenting vessel becomes affected withsluggishness there isusually a sudden appearance of the disease in agreat number of other vessels.

The slightest contact of any portion of a sluggish mash with anothermash is sufiicient to induce sluggishness. If a sample of ebutyl-acetonic bacilli. The precise cirsluggish fermenting mash isfiltered, first through a Buechner filter to remove all solids and thenthrough a Berkefeld filter to remove bacteria, one drop of the filtrateplaced in normal fermenting mash is sufficient to induce sluggishness.Indeed, if one drop of the filtrate is diluted one million times,'a dropof the resultant diluted liquid will frequently induce slugglsh'ness ina normally fermcnt ing mash. "kewise the sluggishness may be propagatedindefinitely by transferring onedrop of the Berkefeld filtrate from a"mash undergoing'sluggish fermentation to a fresh mash, fermenting thefreshmash for about twenty-four hours, securing a Berkefeld filtratefrom it, and repeating the cycle. Such transferring does not involvetrue dilution,

the sluggishness whatever its causebecomes as pronounced in the secondmash as it was in the first. I

On the basis of these facts, and on others not reported here, it wouldappear that slug,- gish fermentation is caused by a living organism ofultra-microscopic dimensions. If this is the case it may be either asaprophyte living in association with the butyl-acetonic organism, oritmay be a true parasite or bacteriophage.

I have now butyl-acetonic bacilli may be rendered resistant to epidemicsof sluggishness, whereby a normal yield of solvents is obtainable at alltimes from carbohydrate fermentation without any interference fromepidemics of sluggishness, or, in the language of my theory, quiteregardless of the presence of an ultra-virus or bacteriophage in themash. I

achieve, this end by immunizing cultures of discovered a process wherebythe butyl-acetonic bacillus tothe presence of example, of 6% corn mashis prepared and inoculated with a spore culture of butyl-acetonicbacilli. It is then heat-shocked for three minutes at 100 0., afterwhich there 1s added a small portion, one or more drops, of clear liquidobtained from acarbohydrate mash undergoing a sluggish butyl-acetomcfermentation by filtering the mash first to remove all solids and secondthrough a Berkefeld filter to remove bacteria. Fermentation is thenallowed to proceed at a temperature of about 36 (l'for-four or moredays-i.le., until spores have developed. This spore cal- I ture is thenused to inoculate fresh corn mash which is again heat-shocked for aboutthree minutes at 100 (3., and treated with two' -The second fermentationis allowed to proceed as before and additional transfers with shockingand with filtrate addition are made-that is, the process is repeated. Atthe end of about ten such treatments the culture thus obtained in sporeform at the end of the last treatment will ordinarily be found to beimmunized in respect to sluggish fermentation, and, if not, thedescribed treatment of the culture iscontinue'd until immunityisattained. The extent of immunity is measured by inoculating a sterilemash with the immune culture, adding a few drops of the Berkefeldfiltrate of a sluggish fermentation, and allowing fermentation toproceed. A fermentation giving a normal solvent yield within a normaltime indicates that the culture is completely immunized.

In making suchtests one employs as controls a. normal non-immunizedculture for fermenting a sterile carbohydrate mash, the result beingtaken as an index of yield and of fermentation time, and one alsoemploys a normal non-immunized culture for fermenting a sterilecarbohydrate mash in the presence of filtrate from a sluggishfermentation, the result being compared with the result obtained fromimmunized cultures. I

The improved results attained with immunized cultures are illustrated bythe following tabulation a Average solvent Number of g: Culture employedtreatments 33,3333

given (2111- mm? ture virus" a the end 0! 72 hours a 1 n 24. 2 R. 2 1025. 3 R 1 10 25. 3 R 4 10 23. 2 None 6.8 None 15. 8 None 19. 6 R 1 1726. 3 R 2 17 25. 8 R 3 17 w. 5 R l 17 24. 9 M None 22. 3' S None l8. 5 S1 None 7. 9

r In addition to clearly disclosing the beneficial result attained bythe use of immunized cultures the tabulation also is useful-in showingthat the process of immunization does not transform low-yieldingcultures into highyielding cultures. For example, Culture R 4, whileimmunized and thus capable of producing a normal fermentation in thepresence -of the ultra-virus, gives consistently lower results than itscompanion cultures. If high solvent yields are to be attained, theculture selected to be immunized must be a is the e normal hi h-yieldingculture under optimum fermentation conditions.

It will also be noted that while the control (non-immunized) culturesshown in the tabulation consistently give much lower yields in thepresence of the ultra-virus than immunized cultures, the actual solventyield of individual non-immunized cultures varies with diflerent tests.For example, Culture M gave a yield of 6.8% in one case and 22.3% inanother case. This uncertainness of result is, of course, characteristicof biological processes. The .point of importance is that non-immunizedcultures give lower average yields than immunized cultures.

While in describing my process for immunizing cultures I have statedthat cultures should be heat-shocked for three minutes at. 100 0., itshould be understood that temperatureand duration of heat shocking maybe varied in accordance with well-known bacteriological technique. Thepurpose of heat-shocking is to eliminate vegetative forms of thebacillus, leaving only the spores to germinate. So long as this resultis attained, the precise method of eliminating attenuated vegetativecells, during the immunifrom sluggish fermentation there is produced anormal, rapid, and complete fermentation with a ood solvent yield. This,of course,

ect observed when a laboratory test of the quality of the culture thusobtained is made. Practically, in'manufacturing operations, thebeneficial effect of the use of such a culture is profound.Sluggishfermentation, as such, disappears from the factory.

While the ultra virus believed to causesluggishness may still haverecurrent periods of activity such that extreme sluggish fermentationwould-occur if ordinary cultures were used, when immunized (resistant)cultures are employed there is no period of sluggish fermentationactually experienced in manufacturing operatibns, and the butylacetonicfermentation process may be operated to consistently produce good yieldsof solvents.

A further very great advantage accrues from the use ofimmunized(resistant) butylacetonic bacilli in conducting the butylacetonicfermentation process-namely that consistently higher yields of solventsare obtained at all times in the manufacturing op erations than areobtained when c rresponding non-immunized cultures are used. Forexample, under normal plant conditions and using non-immunized culturesthe solvent yield, based on dry corn has seldom been over 24%., whenreckoned over an operating period of several months. However, when myimmunized (resistant) cultures are employed,

the solvent yield under plant conditions is maintained consistently at25% or more, based on dry corn. In this connection it should be notedthat generally in the practice ofthe art, solvent yields obtained inlarge scale manufacturing operations are not as great as the best yieldsobtained in laboratory experiments.

There is probably a close connection beu tween the ability of myimmunized (resistant) cultures to produce normal solvent yields frommashes containing the ultra-virus or bacteriophage causing sluggishness,and

their ability to produce higher yields of soll vents in plant practiceat all times, whether or not sluggishness is apparent. 1

Thus, it is possible that in plant practice I the ultra-virus causingsluggishness is almost always present and that actual epidemics ofsluggishness are due to an increase of thevirulence of the ultra-virus.At other times its virulence maybe only suflicient to cause a slightlowering of the final solvent yield obtained fromnon-immunized cultures,without producing the characteristic symptoms of sluggish fermentation.

While I have described the process of immunization as beingpreferentially conducted with some Berlrefeld filtrate from a sluggishfermentation, the filtrate from any other bacterial filter might beemployed. Although any portion ofa sluggishly-fermenting mash might beused to introduce the .virus, the use of unfiltered mash is notprestrains of the bacillus other than the one undergoing treatment, forthe filtrate is not ordinarily obtained from the culture beingimmunized. While I have described specifically the use of corn as acarbohydrate for fermentation it should be pointed out that anyfermentable carbohydrate, saccharine or amylaceous, is useful with myimmunized (resistant) cultures. In the appended claims,

my improved cultures are described as im-' munized, and it should beunderstood that this term is used as synonymous with the term resistant,as was heretofore indicated. IfTow. having described my invention, Icla1m the following as new and novel 1. A process for producing animmunized culture-of butyl-acetonic bacilli which comprises repeatedlysub-cultivating said bacilli .in carbohydrate 'media in the presence ofsome sluggishly-fermenting carbohydrate mash, the cultures beingheat-shocked at each transfer to eliminate attenuated vegetative cells.

2. A process for producing an-immunized culture of butyl-acetonicbacilli which comprises repeatedly sub-cultivating said bacilli incarbohydrate media in the presence of some sluggishly-fermentingcarbohydrate mash, the cultures being shocked forthree ferred, sincesuch procedure will introduce minutes at 100 C. at the time of eachtransfer to eliminate attenuated vegetative cells.

1 3. A process for producing an immunized culture of but l-acetonicbacilli which com;

rises repeate 1y sub-cultivating said bacilli 1n carboh drate media inthe presence of a portion of a Berkefeld filtrate of a carbohydrate mashwhich is undergoing sluggish butyl-acetonic fermentation, the culturesbeing heat-shocked at each transfer to eliminate attenuated vegetativecells.

4. A process for producing an immunized culture ofbutyl-acetonic-b'acilli which comprises sub-cultivating said bacilli tenor more times in a carbohydrate medium in the presence of a portion of aBerkefeld filtrate of a vcarbohydrate mash which is undergoing sluggishbutyl-acetonic fermentation, the cultures being heat-shocked at eachtransfer to eliminate attenuated vegetative cells.

5'. In a process for the production of butyl alcohol and acetone from afermentable carbohydrate mash, the step which comprises inoculating saidmash with an immunized culture of the hereinabove-describedbutylacetonic bacillus.

6. In a process for the production of butyl alcohol and acetone from afermentable amwith butyl-acetonic bacilli, eliminating the attenuatedvegetative cells in said culture, adding to said mash liquid from aslugglshly-fermenting mash, and allowing fermentation'to proceed.

8. In a process for roducing an immunized culture of theliereinabove-described but l-acetonic bacillus for fermentation of a carohydrate mash, the steps which consist in repeatedly subcultivating saidbacilli in carbohydrate media in the presence of somesluggishly-fermenting carbohydrate mash,

the attenuated vegetative cells present in the cultures being eliminatedat each transfer.

9. A process for the. production of butyl alcohol and acetone whichcomprises preparing a sterile'carbohydrate mash, and inoculating saidmash with an immunized culture of the hereinabove-describedbutyl-acetonic bacillus.

10. A process for the production of butyl alcohol and acetonicwhichcomprises preparing a sterile saccharine carbohydrate mash andinoculating said mash with an immunized culture of thehereinabove-described butyl-acetonic bacillus. Y

11. A process for the production of butyl alcohol and acetone whichcomprises inoculating a sterile carbohydrate mash with an immunizedculture of the hereinabove described butyleacetonic bacillus, permittingthe fermentation to go to completion, and re covering the butyl alcoholand acetone from the fermented mash.

I 12. A process for the production of butyl alcohol and acetone whichcomprises preparing a sterile amylaceous mash, and inoculating said mashwith an immunized culture of the hereinabove-described butyl-acetonicbacillus.

13. A" process for the production of butyl alcohol and acetone whichcomprises inoculating a sterile amylaceous mash with an immunizedculture of the hereinabove-described butyl-acetonic bacillus, permittingthe fermentation to go to completion, and recovering the butyl alcoholand acetone 'from the fermented mash.

14. In a process for producing an immunized culture of thehereinabove-described butyl-acetonic bacilli for fermentation of acarbohydrate mash, thesteps which consist in successively subcultivatingand heat shocking said bacilli in the presence of material from asluggishly-fermenting carbohydrate mash.

15. In a process for reducing an immunized culture of theliereinabove-described butyl-acetonic bacilli for fermentation of acarbohydrate mash, the step which consists in cultivating said bacilliin carbohydrate media in the presence of some sluggishly-fermentingcarbohydrate mash.

16. In a process for 1producing an immunized culture of theereinabove-described butyI acetonic bacilli for fermentation of atcarbohydrate mash, the step which consists in cultivating said bacilliin the presence of, a portion of a Berkefeld filtrate of a carbohydratemash which is undergoing sluggish butyl-acetonic fermentation.

In testimony whereof, I aflix my signature.

- DAVID A. LEGG.

